Xenopus laevis as a Model to Identify Translation Impairment

Amélie de Broucker; Pierre Semaille; Katia Cailliau; Alain Martoriati; Thomas Comptdaer; Jean-François Bodart; Alain Destée; Marie-Christine Chartier-Harlin

doi:10.3791/52724

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Method Article

Xenopus laevis as a Model to Identify Translation Impairment

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DOI:

10.3791/52724

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September 27th, 2015

Amélie de Broucker¹ , Pierre Semaille¹ , Katia Cailliau² , Alain Martoriati² , Thomas Comptdaer¹ , Jean-François Bodart² , Alain Destée¹ , Marie-Christine Chartier-Harlin¹

¹Team "Early Stages of Parkinson's Disease" of the Jean-Pierre Aubert Research Center, INSERM UMR-S1172, CHRU Lille, University of Lille 1 and Lille 2, ²Team "Signal Division Regulation", CNRS UMR 8576, University of Lille 1

Summary

Protein synthesis control occurs mainly at the translation initiation step, deficiencies in which are linked to diverse disorders. To better understand their etiology, we described here a protocol using Xenopus laevis oocytes assessing the translation of mos transcript in the presence of a mutant of translation initiation factor eIF4G1.

Abstract

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The initiation step, which involves the assembly of the ribosomal subunits at the mRNA initiation codon, involved initiation factor including eIF4G1. Defects in this rate limiting step of translation are linked to diverse disorders. To study the potential consequences of such deregulations, Xenopus laevis oocytes constitute an attractive model with high degrees of conservation of essential cellular and molecular mechanisms with human. In addition, during meiotic maturation, oocytes are transcriptionally repressed and all necessary proteins are translated from preexisting, maternally derived mRNAs. This inexpensive model enables exogenous mRNA to become perfectly integrated with an effective translation. Here is described a protocol for assessing translation with a factor of interest (here eIF4G1) using stored maternal mRNA that are the first to be polyadenylated and translated during oocyte maturation as a physiological readout. At first, mRNA synthetized by in vitro transcription of plasmids of interest (here eIF4G1) are injected in oocytes and kinetics of oocyte maturation by Germinal Vesicle Breakdown detection is determined. The studied maternal mRNA target is the serine/threonine-protein-kinase mos. Its polyadenylation and its subsequent translation are investigated together with the expression and phosphorylation of proteins of the mos signaling cascade involved in oocyte maturation. Variations of the current protocol to put forward translational defects are also proposed to emphasize its general applicability. In light of emerging evidence that aberrant protein synthesis may be involved in the pathogenesis of neurological disorders, such a model provides the opportunity to easily assess this impairment and identify new targets.

Introduction

Proteins are essential elements of cellular life and thus at larger-scale of the organism. They ensure the majority of cellular functions including structure, transport, reaction catalysis, regulation, gene expression etc. Their expression is the result of a complex mechanism of translation allowing the conversion of an mRNA into protein. The translation is subjected to various controls to adapt and to regulate gene expression according to the cell needs, during development and differentiation, aging, physiological stresses or pathological manifestations.

Translation is divided into 3 phases (initiation, elongation and termination) and....